青兰属黄酮调控Nrf2/ARE信号通路对帕金森病氧化应激损伤的保护作用及机制

Parkinson's disease (PD) is characterized by progressive and selective loss of dopaminergic neurons in substantia nigra pars compacta, for which no effective neuroprotective treatment is currently available. There is increasing evidence showing that oxidative stress is critically involved in the pathogenesis of PD. An extremely promising pathway that confers protection to a variety of oxidative stress-related neurodegenerative insults is the nuclear factor erythroid-2 related factor 2(Nrf2)/antioxidant response element (ARE) pathway. The transcription factor Nrf2 is a key regulator in the coordinated induction of a battery of cytoprotective genes, including those encoding for endogenous antioxidant such as thioredoxin-1 and NQO-1. Our preliminary data showed that many flavonoids isolated from Dracocephalum herbs are robust Nrf2 activators and have potential therapeutic effects targeting PD through activation of the ARE. The objective of the proposal is to investigate whether the flavonoid compounds identified as potent Nrf2 activators can ameliorate Parkinson neurotoxin-induced dopaminergic cell death in cellular and animal models of PD and how Nrf2/ARE pathway and the apoptotic ASK1/JNK pathway are involved. We will use SH-SY5Y dopaminergic cells to examine the protective potential of the Nrf2 activators, and test whether the isolated Nrf2 activators can attenuate nigrostriatal lesions in 6-hydroxydopamine (6-OHDA) induced Parkinson's models in Nrf2-/- and wild-type mice. Finally, we will determine the molecular targets of these compounds. This information will be valuable for future development of new therapeutic strategies for the treatment of Parkinson's disease and other neurodegenerative disorders.

帕金森病（PD）发病机制复杂，目前尚无有效治疗手段，氧化应激可能是多巴胺能神经元变性的最后共同途径。作为细胞抵抗氧化应激损伤最重要的内源性通路，Nrf2/ARE信号通路在PD病理及防治过程中的研究已成为国际上新的热点之一。前期研究发现青兰属多种药用植物中含有的黄酮类成分为强Nrf2诱导剂，可减轻6-OHDA所致SH-SY5Y细胞氧化应激损伤，且此保护作用与Nrf2信号通路诱导密切相关。本课题拟在前期研究基础上系统分离筛选青兰属药用植物中黄酮类Nrf2诱导成分，并通过6-OHDA所致PD动物和细胞模型阐明该类化合物调控Nrf2/ARE通路及下游凋亡相关ASK1/JNK通路对PD氧化应激损伤的保护作用；以活性较强化合物为小分子探针，开展化合物作用机制及靶点研究。本课题将为PD的防治提供一种新策略，同时为筛选新的神经保护药物提供新思路及新途径。

帕金森病是中老年人常见的第二大神经退行性疾病，目前尚无有效治疗措施。氧化应激在帕金森病病理生理过程中占据重要地位。Nrf2-ARE信号通路是体内最重要的内源性抗氧化通路，激活Nrf2-ARE信号通路可能成为帕金森病新的治疗策略。本项目主要完成了青兰属药用植物中黄酮类Nrf2诱导成分的系统分离及活性评价，并对其中Nrf2诱导活性较强的化合物进行了系统的细胞和动物实验，观察了其对6-OHDA所致帕金森病氧化应激损伤的保护作用，并对其作用机制进行了深入的探讨。细胞实验中主要采用6-OHDA损伤SH-SY5Y细胞模型，结果发现化合物可减轻6-OHDA所致氧化应激损伤，表现为细胞存活率增加、LDH漏出率降低、ROS水平的降低及cleaved caspase-3活化的减少，并可同时促进Nrf2及下游靶基因表达，Nrf2 siRNA可阻断化合物的保护作用，提示保护作用由Nrf2信号通路介导。体内试验主要采用6-OHDA立体定位注射法建立小鼠帕金森病动物模型，结果发现化合物可改善阿普吗啡诱导的行为学，降低ROS含量，提高GSH生成，减少黑质纹状体通路DA神经元变性；机制研究发现化合物保护作用与激活Nrf2通路及抑制下游JNK/p38凋亡信号通路有关。课题研究结果系统阐明了该类化合物对PD氧化应激损伤的保护作用及其作用机制，为筛选Nrf2/ARE通路诱导剂作为神经保护剂治疗帕金森病提供了理论基础和开辟了新思路。